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The demand for cheap, healthy, and sustainable alternative protein sources has turned research interest into microbial proteins. Mycoproteins prevail due to their quite balanced amino acid profile, low carbon footprint and high sustainability potential. The goal of this research was to investigate the capability of Pleurotus ostreatus to metabolize the main sugars of agro-industrial side streams, such as aspen wood chips hydrolysate, to produce high-value protein with low cost. Our results indicate that P. ostreatus LGAM 1123 could be cultivated both in a C-6 (glucose)- and C-5(xylose)-sugar-containing medium for mycoprotein production. A mixture of glucose and xylose was found to be ideal for biomass production with high protein content and rich amino acid profile. P. ostreatus LGAM 1123 cultivation in a 4 L stirred-tank bioreactor using aspen hydrolysate was achieved with 25.0 ± 3.4 g L−1 biomass production, 1.8 ± 0.4 d−1 specific growth rate and a protein yield of 54.5 ± 0.5% (g/100 g sugars). PCA analysis of the amino acids revealed a strong correlation between the amino acid composition of the protein produced and the ratios of glucose and xylose in the culture medium. The production of high-nutrient mycoprotein by submerged fermentation of the edible fungus P. ostreatus using agro-industrial hydrolysates is a promising bioprocess in the food and feed industry.

P. ostreatus biomass could stand as an alternative vegan protein source due to its high protein content and amino acid composition.

The ability of the multispecies biofilm membrane reactors (MBM reactors) to provide distinguished niches for aerobic and anaerobic microbes at the same time was used for the investigation of the consolidated bioprocessing of cellulose to short chain fatty acids (SCFAs). A consortium based consolidated bioprocess (CBP) was designed. The rumen microbiome was used as the converting microbial consortium, co-cultivated with selected individual aerobic fungi which formed a biofilm on the tubular membrane flushed with oxygen. The beneficial effect of the fungal biofilm on the process yields and productivities was attributed to the enhanced cellulolytic activities compared with those achieved by the rumen microbiome alone. At 30 °C, the MBM system with Trichoderma reesei biofilm reached a concentration 39% higher (7.3 g/L SCFAs), than the rumen microbiome alone (5.1 g/L) using 15 g/L crystalline cellulose as the substrate. Fermentation temperature was crucial especially for the composition of the short chain fatty acids produced. The temperature increase resulted in shorter fatty acids produced. While a mixture of acetic, propionic, butyric, and caproic acids was produced at 30 °C with Trichoderma reesei biofilm, butyric and caproic acids were not detected during the fermentations at 37.5 °C carried out with Coprinopsis cinerea as the biofilm forming fungus. Apart from the presence of the fungal biofilm, no parameter studied had a significant impact on the total yield of organic acids produced, which reached 0.47 g of total SCFAs per g of cellulose (at 30 °C and at pH 6, with rumen inoculum to total volume ratio equal to 0.372). Electronic supplementary material The online version of this article (10.1007/s00253-019-09706-1) contains supplementary material, which is available to authorized users.

In many plant species, the acetate fraction is the fourth most prominent fraction by weight after cellulose, hemicellulose and lignin, and can be easily extracted as a single stable molecule, acetic acid, at high yields. Despite this, upgrading the acetate fraction of biomass has received very limited attention. Here, we demonstrate a valorization route for the acetate fraction as well as mixtures of acetic acid and other volatile fatty acids produced from the polysaccharide fraction. Aqueous solutions of acetic acid, including solutions produced during steam explosion pretreatment and subsequently purified can be upgraded at high selectivity to a valuable mixture of aromatics, substituted cycloalkenes and gas olefins in a single step using Cu/ZrO2. The catalyst displays remarkable stability despite the presence of acids, water and other biomass-derived impurities. We also show that acetic acid can be further valorized over the same catalyst by converting it in the presence of butanoic acid that was produced in a consolidated bioprocess from the same pretreated wood that was the source of the acetic acid. In this case, the acetic acid rapidly ketonizes with the butanoic acid and the resulting beta-ketones further condense to form aromatics and cycloalkenes with a higher average carbon number than those produced solely from acetic acid. Overall, our process yields a biomass-derived organic oil consisting of aromatics and cycloalkenes that spontaneously separates from water, can be tuned by varying the incoming mixture of carboxylic acids and has suitable properties for being used as a direct blend with aviation fuel.

In this paper, two ethanol processes integrated with a softwood pulp mill are compared with regard to their steam demand, process integration potential and profitability. The processes differ in the solids loading in the simultaneous saccharification and fermentation step and in the resulting ethanol concentration. The results show that a higher ethanol concentration does not necessarily lead to significant reductions in steam demand. Instead, it is demonstrated that the steam demand for distillation is highly dependent on the design of the distillation plant. Nevertheless, a higher solids loading (high gravity) can be beneficial for the treatment of the stillage from the distillation plant. A higher solids loading results either in a lower steam demand for evaporation of the stillage or possibly in a reduced demand for effluent treatment compared to a conventional solids loading process. While the results show that a higher ethanol concentration leads to advantages in energy costs and investment costs for the distillation plant, they also show that the potential benefits of a high-gravity process are offset by the expected decrease in ethanol yield, which leads to higher raw material costs. (C) 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

A co-fermentation process involving Saccharomyces cerevisiae and Fusarium oxysporum was studied, using hydrothermally pretreated wheat straw as substrate. In the first step of the study, we examined liquefaction of the material in a free-fall reactor. Both the enzyme loading and the dry matter content affected severely the liquefaction efficiency. In the second step (simultaneous saccharification and fermentation (SSF) experiments), we found that the enzymatic system of F. oxysporum contributed significantly to substrate hydrolysis, while its metabolic system played a secondary role in fermentation. SSF in the presence of F. oxysporum cells and enzymes gave 62 g L −1 ethanol. In the third step of the study, a semi-consolidated bioprocess was designed in which F. oxysporum culture (submerged or solid-state) was added at the SSF stage along with S. cerevisiae . The addition of solid F. oxysporum culture increased ethanol production by 19%, leading to a final ethanol concentration of 58 g L −1 . The present study proposes a semi-consolidated process combining two microorganisms for the fermentation at high solids concentration of a liquefied material using an in house free fall mixing reactor. The semi-consolidated process proposed not only increased the ethanol yields significantly, but could also lead to lower overall cost of the process by incorporating in-situ enzyme production.

High-gravity (HG) technology aims at generating final ethanol concentrations above 50 kg m(-3) in order to reduce the cost of the distillation step. The generation of higher amounts of inhibitors during the pretreatment step is one of the challenges that accompany the increase in initial dry matter. Detoxification of spruce hydrolysate, adaptation of the cells before fermentation, supplementation with nutrients, and washing of solids were the strategies compared in this study. They represent different approaches to cope with the inhibitory effects, and we compared their efficiencies using a thermotolerant strain of Saccharomyces cereuisiae at temperatures from 30 degrees C up to 40 degrees C. The dilute acid-pretreated spruce used as substrate in this study was not fermentable under HG conditions (200 g kg(-1) water-insoluble solids) when no improvement method was applied. In HG simultaneous saccharification and fermentation at 30 degrees C combined with a 24 h pre-hydrolysis step, the detoxification of pretreated spruce with reducing agent (Na2S2O4) gave the best result with an ethanol yield of 57% (on total sugars) of the maximum theoretical and a volumetric productivity of 1.58 g dm(-3) h(-1). In HG separate hydrolysis and fermentation, nutrients supplementation gave better final ethanol yields than detoxification of the material, reaching an ethanol yield of about 60% of the theoretical (on total sugars). The results obtained, showed an increase in severity of inhibitory effects with temperature increase. Improved cell viability was observed when detoxified material was used and also when yeast extract addition was coupled with adaptation of the cells to the hydrolysate. (C) 2014 Published by Elsevier Ltd.

Background Development of more sustainable biofuel production processes is ongoing, and technology to run these processes at a high dry matter content, also called high-gravity conditions, is one option. This paper presents the results of a life cycle assessment (LCA) of such a technology currently in development for the production of bio-ethanol from spruce wood chips. Results The cradle-to-gate LCA used lab results from a set of 30 experiments (or process configurations) in which the main process variable was the detoxification strategy applied to the pretreated feedstock material. The results of the assessment show that a process configuration, in which washing of the pretreated slurry is the detoxification strategy, leads to the lowest environmental impact of the process. Enzyme production and use are the main contributors to the environmental impact in all process configurations, and strategies to significantly reduce this contribution are enzyme recycling and on-site enzyme production. Furthermore, a strong linear correlation between the ethanol yield of a configuration and its environmental impact is demonstrated, and the selected environmental impacts show a very strong cross-correlation (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r^2>0.9$$\end{document}r2>0.9 in all cases) which may be used to reduce the number of impact categories considered from four to one (in this case, global warming potential). Lastly, a comparison with results of an LCA of ethanol production under high-gravity conditions using wheat straw shows that the environmental performance does not significantly differ when using spruce wood chips. For this comparison, it is shown that eutrophication potential also needs to be considered due to the fertilizer use in wheat cultivation. Conclusions The LCA points out the environmental hotspots in the ethanol production process, and thus provides input to the further development of the high-gravity technology. Reducing the number of impact categories based only on cross-correlations should be done with caution. Knowledge of the analyzed system provides further input to the choice of impact categories. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0468-3) contains supplementary material, which is available to authorized users.

Second-generation bioethanol produced from various lignocellulosic materials, such as wood, agricultural, or forest residues, has the potential to be a valuable substitute for, or a complement to, gasoline. At least three major factors—rapidly increasing atmospheric CO2 levels, dwindling fossil fuel reserves, and their rising costs—suggest that we now need to accelerate research plans to make greater use of plant-based biomass for energy production and as a chemical feedstock as part of a sustainable energy economy. Optimizing the production of bioethanol to be competitive with petrochemical fuels is the main challenge for the underlying process development. The exhaustive research on enzyme technology during the latest years, resulting in significant advances in the field, show the importance of the enzymatic hydrolysis for a profitable ethanol production process. On the other hand, the persisting challenges in biomass pretreatment, which are the initial steps in most process designs, show the remarkable recalcitrance of the lignocellulosic materials to biological degradation. The recent scientific trends show toward an integrated overall bioconversion process in which fermentation technology and genetic engineering of ethanologenic microorganisms aim not only at maximizing yields and productivities but also at widening the range of fermentation products and applications.

BACKGROUND: The high crystallinity of cellulose underlies the recalcitrance that this polymer presents in enzymatic degradation. Thus, a pre-treatment step is applied in most bioconversion processes. Treatments with ionic liquids are considered an emerging pre-treatment technology, owing to their high efficiency in solvating cellulose, over molecular solvent systems. RESULTS: Crystalline cellulose with and without ionic liquid (1-ethyl-3-methylimidazolium acetate) treatment, both commercially available, were used as substrates in enzymatic hydrolysis reactions using the earlier evaluated cellulolytic system of Fusarium oxysporum. The in situ removal of the hydrolysate during reactions enhanced the reaction rate as well as the overall glucose production. Ionic liquid treatment significantly decreased cellulose crystallinity and enhanced bioconversion yields and rates. The effects of cellulose structural changes during treatment on hydrolysis rate were investigated and the recalcitrance constants were determined. CONCLUSION: The study showed that ionic liquid-treated cellulose became more homogeneous and more easily degradable than the untreated cellulose, a conclusion that was expressed mathematically by the difference in the recalcitrance constants for the two substrates. It was concluded that glucose production from ionic liquid-treated cellulose could achieve very high conversion yields in consolidated bioprocesses or during simultaneous saccharification and fermentation. Copyright © 2012 Society of Chemical Industry

The enzymatic degradation of polysaccharides to monosaccharides is an essential step in bioconversion processes of lignocellulosic materials. Alkali treated brewers spent grain was used as a model substrate for the study of cellulose and hemicellulose hydrolysis by Fusarium oxysporum enzyme extract. The results obtained showed that cellulose and hemicellulose conversions are not affected by the same factors, implementing different strategies for a successful bioconversion. Satisfactory cellulose conversion could be achieved by increasing the enzyme dosage in order to overcome the end-product inhibition, while the complexity of hemicellulose structure imposes the presence of specific enzyme activities in the enzyme mixture used. All the factors investigated were combined in a mathematical model describing and predicting alkali treated brewers spent grain conversion by F. oxysporum enzyme extract.

BACKGROUND: During the pretreatment of lignocellulosic materials, molecules such as carboxylic acids, furan derivatives and phenolic compounds, which inhibit the growth and ethanol fermentation by bacteria, fungi and yeasts, are produced. The present work determines the tolerance levels of the C5, C6 fermenting fungus Fusarium oxysporum, towards individual model inhibitory compounds on aerobic growth, on lignocellulolytic activities and on fermentative performance. RESULTS During the growth stage, maximum biomass production was more affected than the specific growth rate by the presence of inhibitors. The presence of high concentrations of inhibitors resulted, in most cases, in prolongation of the lag phase. The fermentative performance of F. oxysporum was significantly inhibited by carboxylic acids, while the lignocellulolytic activities were affected to a lesser extent. CONCLUSION: The toxicity tolerance of fF. oxysporum was high enough for aerobic growth in the presence of significant concentrations of inhibitors, which in most cases were higher than those generated from various treatments of lignocellulosic materials, while its fermentative performance was relatively more affected by the presence of inhibitors. The decrease of ethanol production in the presence of weak organic acids could be the main obstacle to the application of F. oxysporum in large-scale bioconversion processes of hydrolysates containing inhibitors. Copyright © 2010 Society of Chemical Industry

Brewer's spent grain (BG), the most abundant brewing by-product, is used in the present study as a low-cost feedstock for the production of ethanol by the mesophilic fungus Fusarium oxysporum using a consolidated bioconversion process. The production of required cellulolytic and hemicellulolytic enzymes was optimized under solid-state cultivation (SSC) concerning carbon source and initial moisture. The optimal medium contains BG and corn cobs (CC) in a ratio 7:3 while the optimal initial moisture is 66% (w/w). SSC in a laboratory horizontal bioreactor using the optimized medium allowed the large-scale production of a multienzymic system including endoglucanase, cellobiohydrolase, β- d -glucosidase, xylanase, feruloyl esterase, acetyl esterase, β- d -xylosidase and α- l -arabinofuranosidase. Chromogenic (fluorogenic) 4-methylumbelliferyl substrates were used to partially characterize the extracellular proteome of the microbe after the separation by isolectric focusing (IEF) electrophoresis. Alkali pretreatment of brewer's spent grain and different aeration levels were studied for the optimization of the ethanol production by F. oxysporum in a consecutive submerged fermentation. A yield about 65 g ethanol kg−1 of dry BG was obtained with alkali pretreated BG under microaerobic conditions (0.01 vvm) corresponding to 30% of the theoretical yield based on total glucose and xylose composition of BG.

In this study, the ethanol production by the mesophilic fungus Neurospora crassa from BG was studied and optimized concerning the induction of lignocellulose degrading enzymes and the production phase as well. The production of cellulolytic and hemicellulolytic enzymes was studied under solid-state cultivation (SSC). SSC in a laboratory horizontal bioreactor using the optimized medium, WS and BG in the ratio 1:1 and initial moisture level 61.5%, allowed the large scale production of the multienzymatic system. Similar yields with those from flasks experiments, as high as 1073,56,4.2,1.6,3.1,5.7 and 0.52 U g(-1) carbon source of xylanase, endoglucanase, cellobiohydrolase, beta-glucosidase, alpha-l-arabinofuranosidase, acetyl esterase and feruloyl esterase, respectively, were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl substrates were used to characterize the major activities of the multienzyme component, after the separation by isoelectric focusing (IEF) electrophoresis. Alkali pre-treated BG was used for ethanol production. A yield of about 74 g of ethanol kg(-1) dry BG (5,6 g L(-1)) was obtained under optimum conditions (aeration 0.1 vvm, pre-treatment with 1g NaOH 10 g(-1)dry BG).

Background Fusarium oxysporum is a filamentous fungus which has attracted a lot of scientific interest not only due to its ability to produce a variety of lignocellulolytic enzymes, but also because it is able to ferment both hexoses and pentoses to ethanol. Although this fungus has been studied a lot as a cell factory, regarding applications for the production of bioethanol and other high added value products, no systematic study has been performed concerning its ethanol tolerance levels. Results In aerobic conditions it was shown that both the biomass production and the specific growth rate were affected by the presence of ethanol. The maximum allowable ethanol concentration, above which cells could not grow, was predicted to be 72 g/L. Under limited aeration conditions the ethanol-producing capability of the cells was completely inhibited at 50 g/L ethanol. The lignocellulolytic enzymatic activities were affected to a lesser extent by the presence of ethanol, while the ethanol inhibitory effect appears to be more severe at elevated temperatures. Moreover, when the produced ethanol was partially removed from the broth, it led to an increase in fermenting ability of the fungus up to 22.5%. The addition of F. oxysporum’s system was shown to increase the fermentation of pretreated wheat straw by 11%, in co-fermentation with Saccharomyces cerevisiae. Conclusions The assessment of ethanol tolerance levels of F. oxysporum on aerobic growth, on lignocellulolytic activities and on fermentative performance confirmed its biotechnological potential for the production of bioethanol. The cellulolytic and xylanolytic enzymes of this fungus could be exploited within the biorefinery concept as their ethanol resistance is similar to that of the commercial enzymes broadly used in large scale fermentations and therefore, may substantially contribute to a rational design of a bioconversion process involving F. oxysporum. The SSCF experiments on liquefied wheat straw rich in hemicellulose indicated that the contribution of the metabolic system of F. oxysporum in a co-fermentation with S. cerevisiae may play a secondary role.

In brewing and ethanol-based biofuel industries, high-gravity fermentation produces 10–15% (v/v) ethanol, resulting in improved overall productivity, reduced capital cost, and reduced energy input compared to processing at normal gravity. High-gravity technology ensures a successful implementation of cellulose to ethanol conversion as a cost-competitive process. Implementation of such technologies is possible if all process steps can be performed at high biomass concentrations. This review focuses on challenges and technological efforts in processing at high-gravity conditions and how these conditions influence the physiology and metabolism of fermenting microorganisms, the action of enzymes, and other process-related factors. Lignocellulosic materials add challenges compared to implemented processes due to high inhibitors content and the physical properties of these materials at high gravity.

In this study, the factors affecting ferulic acid (FA) release from Brewer’s spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g−1 dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g−1 dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.

Background Brewer's spent grain (BG), a by-product of the brewing process, is attracting increasing scientific interest as a low-cost feedstock for many biotechnological applications. BG in the present study is evaluated as a substrate for lignocellulolytic enzyme production and for the production of ethanol by the mesophilic fungus Fusarium oxysporum under submerged conditions, implementing a consolidated bioconversion process. Fermentation experiments were performed with sugar mixtures simulating the carbohydrate content of BG in order to determine the utilization pattern that could be expected during the fermentation of the cellulose and hemicellulose hydrolysate of BG. The sugar mixture fermentation study focused on the effect of the initial total sugar concentration and on the effect of the aeration rate on fermenting performance of F. oxysporum. The alkali pretreatment of BG and different aeration levels during the ethanol production stage were studied for the optimization of the ethanol production by F. oxysporum. Results Enzyme yields as high as 550, 22.5, 6.5, 3225, 0.3, 1.25 and 3 U per g of carbon source of endoglucanase, cellobiohydrolase, β-D-glucosidase, xylanase, feruloyl esterase, β-D-xylosidase and α-L-arabinofuranosidase respectively, were obtained during the growth stage under optimized submerged conditions. An ethanol yield of 109 g ethanol per kg of dry BG was obtained with alkali-pretreated BG under microaerobic conditions (0.01 vvm), corresponding to 60% of the theoretical yield based on total glucose and xylose content of BG. Conclusion The enzymatic profile of the extracellular extract from F. oxysporum submerged cultures using BG and corn cob as the carbon source was proved efficient for a successful hydrolysis of BG. The fermentation study carried out using sugar mixtures simulating BG's carbohydrates content and consecutively alkali-pretreated and untreated BG, indicates that BG hydrolysis is the bottleneck of the bioconversion process. However, a considerable bioconversion yield was achieved (60% of the theoretical) making this bioprocess worthy of further investigation for a potential commercial application.